Apparent Km For Na Research Articles

Ionomycin, a Ca ++ ionophore, increases platelet volume independently of the [formula omitted] exchanger

A Ca ++-ionophore, ionomycin, increased the volume of human platelets suspended in a Ca ++-containing buffer. This change in cell volume was dependent upon ionomycin and extracellular Ca ++ concentrations, suggesting that the volume change occurs when the intracellular Ca ++ reaches a certain level (>10 uM as determined by aequorin method). The ionomycin-induced volume increase was suppressed by replacement of extracellular Na + with membrane-impermeable N-methyl-D-glucamine or Cs +, but not with Li +, K +, or Rb +. Ethylisopropylamiloride, a potent inhibitor of the Na + H + exchanger, had only weak inhibitory effect, and the apparent Km for Na + was approximately 350 mM, which is much larger than that of the Na + H + exchanger. It is suggested that certain mechanisms other than the Na + H + exchanger are responsible for ionomycin-induced volume increase.

Activation of the Na+/H+ exchanger by phorbol ester and osmotic shock is dependent on the degree of neutrophilic maturation

Activation of neutrophils leading to superoxide production is accompanied by cytoplasmic alkalinization, which results from stimulation of the Na+/H+ exchanger. Since the exchanger undergoes permanent alterations during neutrophilic maturation of HL-60 cells (Costa-Casnellie et al.: Journal of Biological Chemistry 263:11851-11855, 1988), we investigated whether its response to external stimuli such as phorbol esters or osmotic shock also was modified during cell maturation. Mature HL-60 cells produce superoxide in response to active phorbol esters, whereas immature HL-60 cells do not. Stimulation of the exchanger by active phorbol esters (phorbol 12-myristate 13-acetate or phorbol 12,13-dibutyrate) was observed in mature neutrophilic HL-60 cells but not in their immature counterparts. Inactive 4-alpha phorbol had no effect in either cell population. Compound H7 inhibited phorbol ester activation by 65%. In mature neutrophilic cells activation of the exchanger by phorbol esters caused two novel changes of its properties: 1) its apparent Km for Na+ transport increased 2-fold; 2) its Vmax increased 2.6-fold. Phorbol esters also caused a shift in pH dependence of activation similar to that induced in other cells. Osmotic shock, a different method known to activate the exchanger of other cells, induced activation in mature neutrophilic cells but not in immature cells. Thus, the response of the exchanger to external stimuli is affected by alterations occurring in association with cell maturation.

Kinetic properties of the sodium/hydrogen ion antiport of heart mitochondria

The fluorescence of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) has been used to follow the Na+/H+ antiport activity of isolated heart mitochondria as a Na+-dependent extrusion of matrix H+. The antiport activity measured in this way shows a hyperbolic dependence on external Na+ or Li+ concentration when the external pH (pHo) is 7.2 or higher. The apparent Km for Na+ decreases with increasing pHo to a limit of 4.6 mM. The Ki for external H+ as a competitive inhibitor of Na+/H+ antiport averages 3.0 nM (pHo 8.6). The Vmax at 24 degrees C is 160 ng ion of H+ min-1 (mg of protein)-1 and does not vary with pHo. Li+ reacts with the antiporter with higher affinity, but much lower Vmax, and is a competitive inhibitor of Na+/H+ antiport. The rate of Na+/H+ antiport is optimal when the pHi is near 7.2. When pHo is maintained constant, Na+-dependent extrusion of matrix H+ shows a hyperbolic dependence on [H+]i with an apparent Km corresponding to a pHi of 6.8. The Na+/H+ antiport is inhibited by benzamil and by 5-N-substituted amiloride analogues with I50 values in the range from 50 to 100 microM. The pH profile for this inhibition seems consistent with the availability of a matrix binding site for the amiloride analogues. The mitochondrial Na+/H+ antiport resembles the antiport found in the plasma membrane of mammalian cells in that Na+, Li+, and external H+ appear to compete for a common external binding site and both exchanges are inhibited by amiloride analogues.(ABSTRACT TRUNCATED AT 250 WORDS)

A sodium/proton antiporter in chromaffin-granule membranes

Chromaffin granules, the secretory vesicles of the adrenal medulla, have a Na+/H+ exchange activity in their membranes which brings their proton gradient into equilibrium with a Na+ gradient. This explains why Na+ is mildly inhibitory to amine transport (which is driven by the H+ gradient) The activity can be demonstrated by using accumulation of 22Na+ in response to a pH gradient that is either imposed by diluting membrane 'ghosts' into alkaline media, or generated by ATP hydrolysis. It can also be monitored indirectly by fluorescence measurements in which the pH inside 'ghost' is monitored by quenching of a fluorescent weak base. This method has been used to monitor Na+ entry into acid-loaded 'ghosts' of H+ entry into methylamine accumulation. The exchanger appears to be reversible and non-electrogenic, with a stoichiometry of 1:1. Using an indirect assay we measured an apparent Km for Na+ of 4.7 mM, and a Ki for amiloride, a competitive inhibitor, of 0.26 mM. Direct assays using 22Na+ suggested a higher Km. Ethylisopropylamiloride was not inhibitory.

Na+-coupled sugar transport: membrane potential-dependent Km and Ki for Na+.

Kinetic analysis of the characteristics of phlorizin binding and of the Na+, sugar, and potential dependence of alpha-methylglucoside (alpha-MG) influx into isolated avian intestinal cells has pointed toward two alternative models for the transport mechanism (D. Restrepo and G. A. Kimmich, J. Membr. Biol. 89: 269-280, 1986). One of these models envisions a potential-dependent Na+ binding event (Na+ well concept) as a part of the molecular mechanism. The data reported here show that the apparent Km for Na+ for sugar transport is sharply dependent on the magnitude of the membrane potential. When intracellular Na+ is absent, the maximal velocity (Vmax) achieved for sugar influx is the same with or without a potential, although Vmax is obtained at a lower Na+ concentration when a potential is imposed (interior negative). Intracellular Na+ severely inhibits the influx of sugar in the absence of a potential, but this effect is largely overcome when a potential is present. The Vmax obtained when intracellular Na+ is present is a function of the potential. These results are consistent with a transport model in which Na+ binding to the Na+-dependent sugar carrier at the extracellular surface of the membrane and debinding at the inner surface of the membrane are both potential-dependent events.

Kinetic properties of Na+/H+ exchange in cultured bovine pigmented ciliary epithelial cells.

Uptake studies with 22Na were performed in cultured bovine pigmented ciliary epithelial cells, in order to characterize mechanisms of Na+ transport. A large part of Na+ uptake was sensitive to amiloride, quinidine and harmaline. Na+ uptake was stimulated by intracellular acidification (using the NH+4 prepulse technique), and was inhibited with increasing extracellular proton concentration. Decreasing extracellular pH from 7.5 to 7.0 increased the apparent KM for Na+ from 38 to 86 mM without considerable changes in Vmax. In the presence of 5 mM Na+ half maximal inhibition of amiloride sensitive Na+ uptake by extracellular protons was observed at a hydrogen concentration of 50 nM. In the presence of 50 mM Na+ the proton concentration necessary for 50% inhibition was 139 nM. Thus, the mode of inhibition of extracellular H+ seemed to be competitive with a Ki of 20-40 nM. 10 microM amiloride increased the apparent KM for Na+ from 33 mM to 107 mM, while Vmax remained nearly unchanged. IC50 for amiloride was 6 microM at 5 mM Na+ and 36 microM in the presence of 150 mM Na+. Thus, amiloride behaves as a competitive inhibitor with a Ki of about 5 microM. The affinities of Na+ to the transport site (KM approximately 16 mM), to the inhibitory site for protons (KM approximately 21 mM), and to the inhibitory site for amiloride (KM approximately 26 mM) were in the same order of magnitude. In summary, we have presented evidence for the presence of a Na+/H+ exchanger in cultured bovine pigmented ciliary epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Energetic mechanism of system A amino acid transport in normal and transformed mouse fibroblasts.

Ouabain treatment (0.4 mM) of normal and transformed C3H-10T1/2 cells caused a progressive increase in 2-aminoisobutyrate (AIB) transport reaching a maximum after 16 to 18 h exposure. There was a virtually complete blockage of this stimulated rate when 3 microM cycloheximide (CHX) was added together with ouabain at T = 0. In the transformed cell, addition of CHX after 14 h had no effect; in the normal cell, it inhibited (ca. 50%) the final AIB transport rate achieved after 24 h. The t1/2 for reaching maximal activity (insensitive to CHX exposure) was thus shifted from 8 h in the transformed cell to 15 h in the normal cell. Since the rate of achieving maximal activity in the absence of CHX was about the same in the two cells, the shift in t1/2 in the presence of CHX suggests that the rate of degradation is more rapid in the normal cell. Following ouabain treatment, the apparent Km for Na+ was decreased in both cells. The Km returned to the basal level 1 h after ouabain removal in the normal cell, but remained low in the transformed cell during this time period. The stimulation of AIB transport following ouabain removal was largely abolished by a proton ionophore (1799), a lipophilic cation (tetraphenyl-phosphonium), or ouabain. These results suggest that, under the conditions of ouabain stress, there is a switch in the bioenergetic mechanism. The Na+/K+ pump and System A transporter appear to be linked and the membrane potential generated by the Na+/K+ pump activity becomes a major driving force for AIB uptake.

Ursodeoxycholate stimulates Na+-H+ exchange in rat liver basolateral plasma membrane vesicles.

Na+:H+ and Cl-:HCO3- exchange are localized, respectively, to basolateral (blLPM) and canalicular (cLPM) rat liver plasma membranes. To determine whether these exchangers play a role in bile formation, we examined the effect of a choleretic agent, ursodeoxycholate (UDCA), on these exchange mechanisms. 22Na (1 mM) and 36Cl (5 mM) uptake was determined using outwardly directed H+ and HCO3- gradients, respectively. Preincubation of blLPM vesicles with UDCA (0-500 microM) resulted in a concentration-dependent increase in initial rates of amiloride-sensitive pH-driven Na+ uptake, with a maximal effect at 200 microM. UDCA (200 microM) increased Vmax from 23 +/- 2 (control) to 37 +/- 7 nmol/min per mg protein; apparent Km for Na+ was unchanged. Preincubation with tauroursodeoxycholate (200 microM), taurocholate (10-200 microM) or cholate, chenodeoxycholate, or deoxycholate (200 microM) had no effect on pH-driven Na+ uptake. UDCA (200 microM) had no effect on either membrane lipid fluidity, assessed by steady-state fluorescence polarization using the probes 1,6-diphenyl-1,3,5-hexatriene, 12-(9-anthroyloxy) stearic acid, and 2-(9-anthroyloxy) stearic acid (2-AS), or Na+,K+-ATPase activity in blLPM vesicles. In cLPM vesicles, UDCA (0-500 microM) had no stimulatory effect on initial rates of HCO3(-)-driven Cl- uptake. Enhanced basolateral Na+:H+ exchange activity, leading to intracellular HCO3- concentrations above equilibrium, may account for the bicarbonate-rich choleresis after UDCA infusion.

A sodium-hydrogen exchange system in isolated apical membrane from LLC-PK1 epithelia

We have monitored transmembrane pH gradients using acridine orange fluorescence quenching and traced Na+ flux to study the properties of Na+-H+ exchange in apical membrane vesicles isolated from LLC-PK1 epithelia. The membranes have low conductance for Na+, H+, and K+ ions. An outwardly directed K+ gradient in the presence of valinomycin and carbonyl cyanide p-trifluoromethoxyphenyl hydrazone produced intravesicular acidification. This pH gradient was collapsed by addition of extravesicular Na+ or Li+ ions but not by tetramethylammonium. Amiloride (10(-4) M) inhibited the effect of both Na+ and Li+. An outwardly directed Na+ gradient stimulated H+ influx, which was also inhibited by 10(-4) M amiloride. Membrane short-circuit conditions affected neither Na+ nor H+ flux, consistent with transport mediated by an electroneutral process. The interaction of amiloride and sodium is consistent with noncompetitive inhibition with Ki = 100 +/- 10 microM for amiloride and an apparent Km for Na+ of approximately 20 mM. This finding is in agreement with previous studies of intact LLC-PK1 epithelia but differs from observations in brush-border membrane vesicles isolated from kidney proximal tubule in which competitive and mixed inhibition have been reported. These observed differences can be reconciled if two types of Na+-H+ exchange systems exist along the nephron, one with competitive and the other with noncompetitive inhibition, and if only the latter is expressed in the homogeneous cultured cells.

Kinetic properties of the sodium bicarbonate (carbonate) symport in monkey kidney epithelial cells (BSC-1). Interactions between Na+, HCO-3, and pH.

BSC-1 kidney epithelial cells derived from the African green monkey are known to express a Na+HCO3- symport (Jentsch, T. J., Schill, B. S., Schwartz, P., Matthes, H., Keller, S. K., and Wiederholt, M. (1985) J. Biol. Chem. 260, 15554-15560). In the present work, 4,4-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive 22Na+ uptake into confluent monolayers of BSC-1 is measured in the presence of ouabain (10(-4) M) and amiloride (10(-3) M) to define the interactions between Na+ and HCO3- binding and pH. Dependence of DIDS-sensitive 22Na+ fluxes on either Na+ or HCO3- can be described by Michaelis-Menten kinetics. External apparent Km for HCO3- decreases with increasing Na+ concentration (Km app (HCO3-) = 36 +/- 10, 18 +/- 5, and 9 +/- 3 mM at 20, 45, and 151 mM Na+o, respectively (pHo = 7.4)). Similarly, external apparent Km for Na+ decreases with increasing HCO3- concentration (Km app (Na+) = 73 +/- 22, 28 +/- 8, and 14 +/- 4 mM at 6, 17, and 56 mM HCO3o-, respectively (pHo = 7.4)). Vmax app remains constant within the experimental error. When data are replotted as a function of calculated NaCO3- concentration, they can be approximated by a single Michaelis-Menten equation. DIDS-sensitive uptake at constant Na+ and HCO3- displays a broad pH optimum in the range between 7.2 and 7.6. The data are compatible with the ion pair model in which the transported species, NaCO3-, binds to the transport site with Km = 15.3 +/- 4 microM. However, the data may also be fitted by either a random or ordered bireactant system. Sets of parameters necessary for these fits are given.

Actions of NAD+ on renal brush border transport of phosphate in vivo and in vitro.

Previous studies showed that an increase in NAD+ content in renal cortex in vivo was accompanied by specific inhibition of Na+-dependent inorganic phosphate (Pi) transport across the renal brush border membrane (BBM). Further, in vitro addition of NAD+ to isolated renal BBM vesicles specifically inhibited Na+ gradient-dependent transport of Pi. The present study examined some aspects of the mechanism of this inhibition by NAD+ in vitro and in vivo. When NAD+ was increased in vivo by nicotinamide injection, the apparent Vmax was decreased, but the apparent Km was not different, indicating apparent noncompetitive inhibition. In the presence of 0.3 mM NAD+ added in vitro, the apparent Km for Na+-dependent Pi transport by BBM vesicles was increased, whereas the apparent Vmax was unchanged, indicating apparent competitive inhibition. These changes in apparent Km and apparent Vmax were identical when Pi uptake was measured either at 30-s or at 5-s (the initial rate) incubation times. Inhibition of Pi transport by BBM vesicles in vitro was due primarily to the action of intact added NAD+, although there may be some contribution by isotope dilution due to Pi released from NAD+ by enzymatic hydrolysis. Although in vitro inhibition of Pi transport by added NAD+ was reversed by washing the BBM, the inhibition due to increased NAD+ in vivo persisted after extensive washing of the isolated BBM. The specificity of the inhibitory effect of NAD+ in vivo was indicated by the finding that changes in renal cortical content of ATP or Pi, evoked by loading with glycerol or fructose, did not change BBM transport of Pi.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium-induced conformational changes in the glucose transporter of intestinal brush borders.

Using brush-border membranes prepared from rabbit small intestine by Ca2+ precipitation and KSCN treatment, we have studied the kinetics and conformational changes of the glucose carrier. Na+ behaves as a competitive activator of glucose transport under zero-trans conditions. Phenyl isothiocyanate and fluorescein isothiocyanate (FITC) inhibit Na+-dependent transport in an irreversible but substrate-protectable manner. Vesicles pretreated with phenyl isothiocyanate in the presence of substrates were then selectively labeled at the glucose carrier with FITC. Competition experiments with Na+ and phlorizin or glucose indicated that FITC binds to the glucose site on the carrier. Carrier-bound FITC displays a saturable quenching of fluorescence in the presence of Na+. The K0.5 of the Na+-specific quench is 25 mM, which is similar to the apparent Km for Na+ activation of glucose transport. Two tyrosine group-specific reagents, N-acetylimidazole and tetranitromethane, inhibit glucose uptake and fluorescent quenching in a Na+-protectable fashion. FITC labeled a 75-kilodalton peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a substrate-sensitive manner. We conclude that Na+ binds to the glucose symporter of intestinal brush borders, a 75-kilodalton peptide, and this induces a rapid conformation change in the transporter which increases its affinity for D-glucose.

Saturation characteristics, Ca2+ ions and drug-induced block of cardiac Isi-mediated action potentials.

Isi saturation characteristics and the interaction of Ca2+ with the slow channel blockers verapamil and nifedipine were studied in microelectrode experiments on papillary muscles of guinea pigs by analyzing \(\dot V_{\max } \) of Isi-mediated action potentials. 1. A non-linear response of \(\dot V_{\max } \) on external Ca2+ variations between 2 and 6 mmol/l was obtained strongly suggesting saturation characteristics of the Isi system. Transformation of each individual \(\dot V_{\max } \)-[Ca2+]0 relationship into the Lineweaver-Burk form revealed apparent Km's for Ca2+ which varied from 1.5 to 31.2 mmol/l (mean: 7.96±1.07 mmol/l). Values of 20–108 V/s were calculated for the saturated \(\dot V_{\max } \), i.e. \(\dot V_{\max } \) at an infinitely high [Ca2+]0. 2. Histamine did not alter the apparent Km for Ca2+ but produced a concentration-dependent rise of the saturated \(\dot V_{\max } \). 3. Alkalinization of the medium lowered the apparent Km for Ca2+ from 9.2±1.1 mmol/l (at pH 7.4) to 2.7±0.2 mmol/l (at pH 9.0). 4. Sr2+ and Na+ compete with Ca2+ for common anionic sites. The ratio Ksr2+: KCa2+ amounted to 1.75±0.21. The apparent Km for Na+ should be much smaller than KCa2+ because external Na+ reduction from 150 to 75 mmol/l led to an increase of \(\dot V_{\max } \). 5. Nifedipine and, when analyzed under rested-state conditions, verapamil induced alterations of the \(\dot V_{\max } \)-[Ca2+]0 relationship which perfectly fit the formalism of competitive interaction. The y-intercept of the straight relating 1/\(\dot V_{\max } \) to 1/[Ca2-]0 remained either unchanged or varied insignificantly whilst the x-intercept strongly shifted to higher values. Repetitive stimulation changed the mode of verapamil action from a competitive to a non-competitive type. 6. Ca2+ modulates both the verapamil- and the nifedipine-induced Isi block in that excess Ca2+ attenuates drug action and vice versa. The same decline of block develops after adding Sr2+ to the superfusate. 7. The Ca2+ sensitivity of the both types of varapamil-induced Isi inhibition, tonic (rested-state) and phasic (use-dependent) block, differs considerably. The former declined in response to a threefold increase in external Ca2+ by a factor of 3.18, but the latter (analyzed at a driving rate of 12/min) by a factor of only 1.18. Consistent with this pecularity, concentration-response analysis of the verapamil action yielded individual apparent dissociation constants and nH coefficients being (at 3 mmol/l Ca2+) 6.4×10−6 mol/l and 3.91 for tonic Isi blockade, but 1×10−6 mol/l and 1.06 for phasic (frequency 12/min) Isi blockade.

A quantitative analysis of the Na+-dependence of Vmax of the fast action potential in mammalian ventricular myocardium. Saturation characteristics and the modulation of a drug-induced INa blockade by [Na+]o.

In microelectrode experiments on papillary muscles of guinea pigs, the quantitative dependence of\(\dot V_{{\text{max}}} \) of the fast action potential, taken as a measure of INa, on external Na+ concentration has been analyzed under different experimental conditions including the presence of antiarrhythmic drugs such as lidocaine, procaine and propafenone. 1. External Na+ concentration changes between 225 mmol/l and 45 mmol/l led to a non-linear response of\(\dot V_{{\text{max}}} \) in that the\(\dot V_{{\text{max}}} \) changes obtained experimentally were significantly smaller than predicted theoretically from a linear dependence of\(\dot V_{{\text{max}}} \) on [Na+]o. 2. Each individual\(\dot V_{{\text{max}}} \) relationship exhibited saturation characteristics. In Lineweaver-Burk plots,\(\dot V_{{\text{max}}} \) correlated extremely well to 1/[Na+]o with correlation coefficients between 0.995 and 0.999. In 16 experiments, the apparentKm for Na+ varied within a range from 170 to 455 mmol/l. Values of 450 V/s–900 V/s were calculated for the saturated\(\dot V_{{\text{max}}} \) (at an infinitely large [Na+]o). 3. The apparentKm for Na+ rose from 232.0±24.7 mmol/l to 544.0±50.2 mmol/l when the K+ concentration of the medium was increased from 5.4 to 10 mmol/l and, thus, resting potential declined from −90.5±2.5 mV to −76.1±1.8 mV. 4. Alkalization (pH 9.0) of the medium lowered the apparentKm for Na+ and, simultaneously, reduced the saturated\(\dot V_{{\text{max}}} \). This typical shift of the straight relating\(\dot V_{{\text{max}}} \) to 1/[Na+]o in the Lineweaver-Burk plot excludes a competitive interaction between Na+ and H+ ions. 5. Lidocaine (5×10−5–2×10−4 mol/l), procaine (2×10−4 mol/l) and propafenone (0.5–3×10−5 mol/l) depressed\(\dot V_{{\text{max}}} \) the stronger the lower [Na+]o was. The changes of the\(\dot V_{{\text{max}}} \) relationship induced by these drugs indicated neither a competitive nor a non-competitive interaction of these compounds with Na+. 6. As tested with propafenone, external Na+ changes modulated the tonic and the phasic block of\(\dot V_{{\text{max}}} \). The Na+ sensitivity of both types of block differed considerably. In a Na+-poor (50 mmol/l) medium, the apparentKm for the tonic block declined from 5×10−5 to 1.4×10−5 mol/l and the apparentKm for the phasic block from 3.8×10−5 to 2.3×10−5 mol/l. 7. Na+ is not the sole cation that determines the strength of a drug-induced blockade of\(\dot V_{{\text{max}}} \) as it can be substituted by the permeant Li+. 8. In the presence of drugs like propafenone which are capable of shifting the steady state inactivation (h∞) of INa to more negative potentials, the voltage-dependence ofh∞ can be modified by external Na+ variations. Na+ withdrawal led to a considerable shift in the hyperpolarizing direction.

Amino acid transport in plasma-membrane vesicles from rat liver. Characterization of L-alanine transport.

Plasma-membrane vesicles, isolated from rat liver, catalyze active transport of L-alanine. The transient accumulation of L-alanine requires the presence of Na+ concentration gradient (outside greater than inside). The alanine-Na+ symport is an electrogenic process, since it is stimulated under conditions that allow compensatory ion movements: both co-transported anions as well as counter-transported cations influence the rate of alanine-Na+ symport. However, no uptake is observed in the presence of a membrane potential, when no Na+ concentration gradient is present. Sodium-gradient-stimulated alanine uptake is dependent on temperature and pH, stereospecific, and is affected by the presence of other amino acids. The activity of the L-alanine transport system is influenced both by the Na+ and by the L-alanine concentration. In the presence of 100 mM Na+, an apparent Km for alanine of 2 mM is observed; lowering the Na+ concentration results in an increase in the apparent Km, and a decrease in the apparent V. The apparent Km for Na+ is 34 mM in the presence of 0.2 mM L-alanine. Increasing the L-alanine concentration also results in a lower apparent Km for Na+, and a higher V.

Coupling of opiate receptors to adenylate cyclase: requirement for Na+ and GTP.

Inhibition of the adenylate cyclase activity in homogenates of mouse neuroblastoma-glioma hybrid cells (NG108-15) by the opioid peptide [D-Ala2,Met5]enkephalin amide (AMEA) requires the presence of Na+ and GTP. In this process, the selectivity for monovalent cations is Na+ greater than or equal Li+ greater than K+ greater than choline+; ITP will replace GTP but ATP, UTP, or CTP will not. The apparent Km for Na+ is 20 mM and for GTP it is 1 microM. Under saturating Na+ and GTP conditions, the apparent Ki for AMEA-directed inhibition is 20 nM for basal and 100 nM for prostaglandin E1-activated adenylate cyclase activity. For both cyclase activities, maximal inhibition is only partial (i.e., approximately 55% of control in each case). In intact viable NG108-15 cells, the decrease in basal and prostaglandin E1-stimulated intracellular cyclic AMP concentrations by AMEA is also dependent upon extracellular Na+. The enkephalin-directed reductions in cyclic AMP concentrations are at least 75%. The specificity of the monovalent cation requirement for enkephalin action on intact cells is the same as for enkephalin regulation of homogenate adenylate cyclase activity. Based on these data, a model is presented in which the transfer of information from opiate receptors to adenylate cyclase requires active separate membrane components, which correspond to the sites of action of Na+ and GTP in this process.